Abstract
Heparan sulfate (HS) catabolism begins with endo-degradation of the polysaccharide to smaller HS oligosaccharides, followed by the sequential action of exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate. In mucopolysaccharidosis type IIIA (MPS IIIA) the exo-enzyme, N-sulfoglucosamine sulfohydrolase, is deficient resulting in an inability to hydrolyze non-reducing end glucosamine N-sulfate esters. Consequently, partially degraded HS oligosaccharides with non-reducing end glucosamine sulfate esters accumulate. We investigated the distribution of these HS oligosaccharides in tissues of a mouse model of MPS IIIA using high performance liquid chromatography electrospray ionization-tandem mass spectrometry. Oligosaccharide levels were compared to total uronic acid (UA), which was used as a measure of total glycosaminoglycan. Ten oligosaccharides, ranging in size from di- to hexasaccharides, were present in all the tissues examined including brain, spleen, lung, heart, liver, kidney and urine. However, the relative levels varied up to 10-fold, suggesting different levels of HS turnover and storage. The relationship between the di- and tetrasaccharides and total UA was tissue specific with spleen and kidney showing a different disaccharide:total UA ratio than the other tissues. The hexasaccharides showed a stronger correlation with total UA in all tissue types suggesting that hexasaccharides may more accurately reflect the storage burden in these tissues.
Highlights
Degradation of the glycosaminoglycan (GAG), Heparan sulfate (HS), begins with endo-degradation of the polysaccharide to smaller HS fragments, followed by the sequential action of lysosomal exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate for reutilization by the cell
While changes in GAG and uronic acid (UA) have been reported in MPS IIIA mouse tissues and urine, these methods measure a mixture of oligo- and polysaccharides and do not discriminate between oligosaccharides derived from HS, chondroitin sulfate or dermatan sulfate
A similar distribution of UA was seen in the wild type and MPS IIIA mouse tissues with the kidney showing the highest level (Figure 1)
Summary
Degradation of the glycosaminoglycan (GAG), HS, begins with endo-degradation of the polysaccharide to smaller HS fragments, followed by the sequential action of lysosomal exo-enzymes to reduce these oligosaccharides to monosaccharides and inorganic sulfate for reutilization by the cell. Total urinary GAG is often used as a biochemical measure of disease activity in MPS IIIA using cetylpyrinium chloride and ethanol precipitation of GAG followed by the measurement of free and conjugated UA by the hydroxydiphenol method [2]. While changes in GAG and UA have been reported in MPS IIIA mouse tissues and urine, these methods measure a mixture of oligo- and polysaccharides and do not discriminate between oligosaccharides derived from HS, chondroitin sulfate or dermatan sulfate. Using ESI-MS/MS in combination with enzyme and chemical digestion we identified mono- to hexadecasaccharides in the urine of an MPS IIIA patient [20] These oligosaccharides were composed of N-acetylated (GlcNAc-UA) and unsubstituted glucosamine (GlcN-UA) repeating disaccharides with up to two sulfates per disaccharide. We assessed whether the oligosaccharides detected in the urine reflect a contribution of the oligosaccharides in all tissues or just those present in the kidney
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